類別:

龐貝氏症:新生兒篩檢發現個案之突變分析及治療成果

Thu, 01 Jan 2009 00:00:00 GMT

標題: 龐貝氏症:新生兒篩檢發現個案之突變分析及治療成果; Pompe disease: novel mutations and treatment outcome through newborn screening 作者: Yin-Hsiu Chien; 簡穎秀摘要: 背景: 龐貝氏症是一種因為溶小體缺乏酸性醣苷酶無法分解肝醣所引起之疾病。最近針對龐貝氏症，已有酵素補充療法可以使用。嬰兒型龐貝氏症患者經由酵素補充療法，可有效延長性命，恢復心臟正常大小，但是大部分患者的運動及呼吸功能無法恢復正常；晚發型龐貝氏症治療後，其症狀之可逆性也很有限。本研究的目的在探求新生兒篩檢可以早期診斷龐貝氏症，給予患者早期治療的機會。方法: 我們首先分析台灣龐貝氏症患者之表現型、基因型、以及個案之分佈，以及是否有導致治療失敗之因子。我們接著開發龐貝氏症新生兒篩檢之方法，並且進行前導篩檢作業。篩檢結果之分析，包括篩檢個案之表現型及基因型分析，以及嬰兒型龐貝氏症早期治療之成果。經由比較臨床個案及篩檢結果，試圖去預測台灣族群龐貝氏症之分佈，以及台灣未來關於龐貝氏症之診斷與治療之策略。結果: 臨床個案分析顯示嬰兒型龐貝氏症患者接受酵素治療後，其肌肉對治療之反應不一，但是並沒有證據顯示患者之腦部會有不可逆之變化。經由新生兒篩檢檢出之嬰兒型患者，因為治療開始時間較早，其預後明顯比臨床個案要好。這些患者的基因型與臨床發現個案相類似，主要為p.D645E突變。新生兒篩檢同時檢出部分患者酵素活性缺乏，懷疑為晚發型龐貝氏症患者。這些患者的基因型與臨床發現的晚發型龐貝氏症患者部分類似，但是其發生率以及突變位置未確定的比例均較臨床個案為高。經由族群篩檢我們發現了高比例之龐貝氏症基因變異型。這些變異型會使得酸性醣苷酶活性偏低，我們目前不能排除這些基因變異型是否參與龐貝氏症臨床表現之決定。結論與展望: 新生兒篩檢可以達到早期診斷龐貝氏症之目的，並改善嬰兒型龐貝氏症之預後。台灣族群中帶有相當高比例之龐貝氏症基因變異型，可能引起龐貝氏症診斷之困難或混淆。新生兒篩檢疑似晚發型龐貝氏症之發生率尚待進一步證實，但結果也暗示臨床上可能有部分晚發型龐貝氏症患者沒有被診斷出來。; Background: Pompe disease is due to a deficiency of lysosomal acid alpha glucosidase (GAA), and currently an enzyme replacement therapy has been developed. In infantile-onset Pompe disease, enzyme replacement therapy can prolong survival and reverse cardiomegaly, however, some patients cannot regain motor or respiratory function. In late-onset Pompe disease, most of the symptoms cannot be reversed by treatment. In this study, we hypothesis that newborn screening for Pompe disease can offer an opportunity for early treatment of Pompe disease. Methods: We first analyzed phenotype and genotypes of Pompe disease in Taiwan, and explored factors which may affect the outcome. We then developed a method of newborn screening for Pompe disease, and started a pilot screening program. We analyzed the genotypes, aiming at predicting phenotypes and comparing outcomes between early treatment and late treatment. Results: Patients diagnosed clinical during infancy have poor responses to the treatment in view of skeletal muscle function. However, there was no irreversible changes in their brains. Patients with infantile-onset pompe disease identified by newborn screen could have an earlier onset of treatment and better outcomes in comparism to those identified by clinical symptoms. GAA gene mutations were similar between the two groups, and p.D645E is the most common one. We also identified babies with GAA deficiency but may have late-onset Pompe disease. They had GAA gene mutations previously seen in patients with late-onset Pompe disease, but the babies tended to have a higher incidence of mutations with unknown significance. Through newborn screening, we also identified prominent GAA gene variations in our population, including the “pseudodeficiency” allele and alleles which may modify the clinical manifestations of Pompe disease. Conclusion: Results from this study highlight the benefits of early diagnosis, which can be achieved only by newborn screening. of the intense gene variations in the population can confuse the diagnosis. The high incidence of late-onset Pompe disease from the screening program needs further confirmation, but our results do suggest the possibility of under diagnosis of Pompe disease in current clinical practice.

鼻腔及口腔微生物菌叢在氣喘的角色

Mon, 01 Jan 2024 00:00:00 GMT

標題: 鼻腔及口腔微生物菌叢在氣喘的角色; The role of nasal and oral microbiome in asthma 作者: 張嘉凌; Chia-Ling Chang 摘要: 背景：氣喘是呼吸道對許多過敏原反應過度而發生慢性反覆性的呼吸道發炎疾病。微生物菌叢與呼吸道黏膜上皮細胞緊密接觸，微生物菌叢失調可能是導致呼吸道慢性發炎的重要原因之一。上下呼吸道不論結構上或功能上皆緊密相關，因此，鼻腔及口腔微生物菌叢失調，亦可能對於下呼道慢性發炎，如氣喘的疾病控制，造成顯著的影響。微生物菌叢在氣喘病患急性發作易感性上所扮演的角色。方法：本研究在國立臺灣大學醫學院附設醫院及其新竹分院進行。收案診斷為氣喘的成人病患，並追蹤三個月。所有病患在收案後均接受鼻腔和口腔微生物菌叢的檢查。結果：本研究一共收案61名氣喘病患。病患年齡中位數為44歲（範圍：21–72歲）。男性佔所有病患比例的18%。回歸分析發現：鼻腔細菌叢的alpha多樣性與ACT分數呈負相關和eosinophil數量呈正相關，但與IgE濃度跟FEV1/FVC數值的相關性不顯著。收案後三個月內，34%的病患曾經有氣喘急性惡化。曾經發生氣喘急性惡化病患的鼻腔細菌叢的alpha多樣性(alpha diversity)有低於無急性惡化病患的傾向。兩組間鼻腔細菌叢組成不同。相較於未發生氣喘急性惡化病患，曾經發生急性惡化的患者鼻腔在種的層級有較低比例的Cutibacterium acnes (1.44±2.14% vs. 4.11±6.34%, P = 0.019)、Anaerococcus Octavius (0.06±0.26% vs. 0.54±1.31%, P = 0.029)、 Pseudomonas yamanorum (0.02±0.08% vs. 0.40±0.97%, P = 0.019)、Acinetobacter johnsonii (0.02±0.08% vs. 0.07±0.19%, P = 0.015)、Pseudobdellovibrio exovorus (0.01±0.03% vs. 0.13±0.27%, P = 0.037)和 Brevibacillus marinus (0.002±0.01% vs. 0.05±0.14%, P = 0.032)；在屬的層級有較低比例的Cutibacterium (1.55±2.19% vs. 4.36±6.42%, P = 0.015), Anaerococcus (0.09±0.36% vs. 1.02±2.36%, P = 0.018), Lactobacillus (0.00±0.00% vs. 0.34±0.95%, P = 0.031), Blautia (0.001±0.004% vs. 0.11±0.34%, P = 0.039), Pseudobdellovibrio (0.01±0.03% vs. 0.07±0.19%, P = 0.036), Brevibacillus (0.003±0.01% vs. 0.06±0.13%, P = 0.016)；在門的層級有較低比例的 Cyanobacteria (0.00±0.00% vs. 0.02±0.07%, P = 0.046)。在口腔細菌叢方面，我們發現口腔細菌叢的alpha多樣性與ACT分數、eosinophil數量、IgE濃度和FEV1/FVC數值的相關性不顯著。兩組間，口腔細菌叢的alpha多樣性和beta多樣性皆無顯著差異。然而，相較於未發生氣喘急性惡化病患，曾經發生急性惡化的患者口腔在種的層級有較低比例的Streptococcus sanguis (0.29±0.32% vs. 0.69±1.08%, P = 0.036)、 Streptococcus gwanjuense (0.02±0.05% vs. 0.16±0.43%, P = 0.041)、Actinomyces graevenitzii (0.04±0.06% vs. 0.17±0.38%, P = 0.033)、Butyrivibrio hungatei (0.01±0.02% vs. 0.04±0.06%, P = 0.047)和Leptotrichia buccalis (0.004±0.01% vs. 0.02±0.05%, P = 0.035)；在屬的層級有較低比例的Butyrivibrio (0.02±0.02% vs. 0.04±0.06%, P = 0.048)。結論：曾急性惡化的氣喘病患，其鼻腔細菌叢的alpha多樣性似乎低於未曾氣喘急性惡化病患。曾氣喘急性惡化患者的鼻腔中，在屬的層級有較低比例的Cutibacterium、Anaerococcus、Lactobacillus、Blautia、Pseudobdellovibrio 和 Brevibacillus。口腔細菌叢的alpha多樣性和beta多樣性在曾急性發作與未曾急性發作病患中皆無顯著差異。本研究未收案健康人做比較，且收案人數較少。鼻腔微生物和氣喘急性發作易感性的機轉，需要進一步以動物介入實驗探討。; Background: Asthma is a chronic and recurrent inflammatory disease of the airways, triggered by an overreaction of the respiratory system to various allergens. The microbiome is in close contact with the respiratory mucosal epithelial cells, and its dysbiosis may be one of the key factors contributing to chronic airway inflammation. The upper and lower respiratory tracts are closely related both structurally and functionally. Therefore, dysbiosis of the nasal and oral microbiota may also significantly impact the control of chronic lower airway inflammation, such as in asthma. The role of the microbiome in the susceptibility to acute exacerbations in asthma patients is also noteworthy. Methods: The study conducted at National Taiwan University Hospital in Taipei and its Hsinchu Branch in Taiwan. Adult patients diagnosed with asthma were recruited at 3-month follow-up. All patients underwent nasal and oral microbiome examinations after enrollment. Results: A total of 61 patients with asthma were enrolled. The mean patient age was 44 years (range: 21–72 years). Men comprised 18% (11/61) of all patients. Within the 3-month period, 34% of the patients experienced exacerbations. Patients with exacerbated asthma tended to have lower nasal alpha diversity than those without exacerbations. Nasal alpha diversity may correlate negatively with ACT scores and positively with eosinophil counts, but not with IgE levels or value of FEV1/FVC. The nasal bacterial composition differed between the two groups. Compared to patient with non-exacerbated asthma, those with exacerbated asthma had a lower proportion of nasal Cutibacterium acnes (1.44±2.14% vs. 4.11±6.34%, P value = 0.019), Anaerococcus octavius (0.06±0.26% vs. 0.54±1.31%, P value = 0.029), Pseudomonas yamanorum (0.02±0.08% vs. 0.40±0.97%, P value = 0.019), Acinetobacter johnsonii (0.02±0.08% vs. 0.07±0.19%, P value = 0.015), Pseudobdellovibrio exovorus (0.01±0.03% vs. 0.13±0.27%, P value = 0.037), and Brevibacillus marinus (0.002±0.01% vs. 0.05±0.14%, P value = 0.032) at species level, had a lower proportion of nasal Cutibacterium (1.55±2.19% vs. 4.36±6.42%, P value = 0.015), Anaerococcus (0.09±0.36% vs. 1.02±2.36%, P value = 0.018), Lactobacillus (0.00±0.00% vs. 0.34±0.95%, P value = 0.031), Blautia (0.001±0.004% vs. 0.11±0.34%, P value = 0.039), Pseudobdellovibrio (0.01±0.03% vs. 0.07±0.19%, P value = 0.036), Brevibacillus (0.003±0.01% vs. 0.06±0.13%, P value = 0.016) at the genus level, and had a lower proportion of nasal Cyanobacteria (0.00±0.00% vs. 0.02±0.07%, P value = 0.046) at the phylum level. There were no differences in oral alpha diversity and beta diversity between the two groups. Oral alpha diversity index was not associated with ACT score, eosinophil count, IgE level or value of FEV1/FVC. However, compared with patients with non-exacerbated asthma, those with exacerbated asthma had a lower proportion of oral Streptococcus sanguis (0.29±0.32% vs. 0.69±1.08%, P value = 0.036), Streptococcus gwanjuense (0.02±0.05% vs. 0.16±0.43%, P value = 0.041), Actinomyces graevenitzii (0.04±0.06% vs. 0.17±0.38%, P value = 0.033), Butyrivibrio hungatei (0.01±0.02% vs. 0.04±0.06%, P value = 0.047), and Leptotrichia buccalis (0.004±0.01% vs. 0.02±0.05%, P value = 0.035) at the species level, and had a lower proportion of oral Butyrivibrio (0.02±0.02% vs. 0.04±0.06%, P value = 0.048) at the genus level. Discussion: The nasal microbiome may be an important factor influencing susceptibility to asthma exacerbations. Patients with exacerbated asthma seemed to have lower alpha diversity in their nasal microbiome compared to those with non-exacerbated asthma. Patients with exacerbated asthma had a lower proportion of nasal Cutibacterium, Anaerococcus, Lactobacillus, Blautia, Pseudobdellovibrio, and Brevibacillus at the genus level. There were no significant differences in the alpha diversity and beta diversity of the oral microbiome between patients who had experienced exacerbations and those who had not. This study did not include healthy individuals for comparison, and the sample size was relatively small. Further animal interventional studies are needed to explore the mechanisms by which nasal microbiome may influence susceptibility to asthma exacerbations.

鼻咽癌患者Caveolin-1的表現與臨床特性之研究

Mon, 01 Jan 2007 00:00:00 GMT

標題: 鼻咽癌患者Caveolin-1的表現與臨床特性之研究; Expression of Caveolin-1 and its impact on clinical behavior of nasopharyngeal carcinoma patients 作者: Ying-Piao Wang; 王瀛標摘要: 研究背景: 鼻咽癌原發於鼻咽部上皮細胞，是一種在白種人非常罕見的惡性腫瘤，然而在中國東南沿海省分、香港以及台灣卻是一個常見的疾病，顯示此疾病不同於其它頭頸部癌症，具有種族性、地域性與特殊致病因子。鼻咽癌發生之機轉乃多重因素所構成，目前認為和遺傳因子、環境因素以及Epstein-Barr病毒感染等三大項有關。潛伏期膜蛋白-1(LMP-1)與潛伏期膜蛋白2A(LMP2A)為EB病毒潛伏期表現的產物，在鼻咽癌檢體中65%-95%可測得其轉錄產物的表現，LMP-1是EB病毒使細胞轉型(transformation)的主要蛋白，而LMP2A蛋白可透過活化PI3-kinase-Akt路徑使上皮細胞株細胞轉型增生並抑制細胞分化。根據研究不論是LMP-1或LMP2A都和細胞膜上的脂筏(lipid rafts)相關，Caveolae則是具有調控多種細胞訊息傳遞的一種脂筏，主要的組成蛋白為Caveolin-1，最近有研究指出Caveolin-1可以調控PI3K/Akt路徑進而增強細胞的存活。目前已知Caveolin-1在膀胱癌、食道癌、甲狀腺乳突癌、攝護腺癌及T細胞血癌有一致性升高的趨勢，而在卵巢癌、肺癌、乳癌則為降低的現象，至於鼻咽癌則文獻尚未有報告。研究目的: 本論文嘗試研究台灣地區鼻咽癌組織中Caveolin-1 mRNA的表現量與對照組之比較，其次想探討Caveolin-1的表現量高低和病患臨床表現如年齡、性別、病理分類、血中膽固醇、腫瘤分期、遠處轉移、腫瘤復發及存活率之相關性。最後利用In vitro鼻咽癌細胞株分析Caveolin-1的表現量是否會影響Akt及磷酸化Akt蛋白質的表現量。研究方法: 本研究採用回溯性(retrospective)方式進行分析，收集台大及馬偕醫院鼻咽癌檢體(1990年到2002年間)，及非鼻咽癌鼻咽組織為對照檢體。萃取mRNA，利用同步定量聚合酶連鎖反應分析Caveoin-1 mRNA在不同檢體的表現量。再依上述資料統計分析Caveoin-1在鼻咽癌的表現及其對鼻咽癌患者年齡、性別、病理分類、臨床分期、腫瘤轉移、預後的影響。進而分析鼻咽癌檢體中Caveoin-1和Akt的表現量之相關性。最後以shRNA-Cav1 降低 (knock down)鼻咽癌TW01細胞株中的Caveolin-1 並探討其影響Akt及磷酸化Akt 蛋白質表現量的能力。結果: 初步研究結果顯示，鼻咽癌患者Caveolin-1的表現量比對照組高，且具有統計上的差異(p=0.0002)。進一步分析在鼻咽癌74名患者中，臨床上晚期(AJCC stage III,IV)患者比早期(AJCC stage I,II)患者有較高的Caveolin-1的相對表現量(p=0.0002)。使用回歸分析發現只有臨床分期、原發腫瘤侵犯的範圍(T)、淋巴節轉移情形(N)等因子有顯著統計差異(臨床分期P<0.001，腫瘤侵犯的範圍P=0.017，淋巴節轉移情形 P=0.015)，而年齡、性別、病理WHO分纇、遠端轉移、顱內侵犯均無顯著差異。存活分析則用Kaplan-Meier method，以Caveolin-1的中位數為準，將其分為高、低兩組，Caveoin-1表現較高者存活率較差(Log-rank test，P=0.03)。而在體外試驗中顯示鼻咽癌TW01細胞株中，過度表現Caveolin-1可以增加磷酸化Akt蛋白量﹔而以shRNA降低Cav-1的量會減少磷酸化Akt蛋白以及Akt蛋白的表現。結論: 鼻咽癌患者Caveolin-1的表現量比對照組高，其表現量和臨床分期、原發腫瘤侵犯的範圍(T)、淋巴節轉移情形(N)三個因子有顯著統計差異且Caveoin-1表現較高者鼻咽癌存活率較差。而本實驗亦證明在鼻咽癌TW01細胞株中，Caveolin-1可以調控Akt的表現。; Nasopharyngeal carcinoma (NPC) is a squamous-cell carcinoma occurring in the epithelial lining of the nasopharynx. The incidence is high in Southeast China, Hong Kong and Taiwan, however it is rare in western countries indicating a remarkably distinctive ethnic and geographic distribution that differing from other head and neck cancers. The tumorgenesis of NPC is a multi-factorial process, including genetic, environmental and Epstein-Barr virus. Latent membrane protein 1(LMP-1) and Latent membrane protein 2A (LMP2A) are EBV-encoded proteins expressed during latent phase, and transcripts are detected in 65% and 95% of the tumors respectively. It has been demonstrated EBV-encoded LMP-1 can cause cellular transformation, and LMP2A transforms epithelial cells, inhibits cell differentiation through activating PI3-kinase-Akt pathway. Interesting, both LMP-1 and LMP2A is targeted to lipid rafts/caveolae. Caveolin-1, an essential protein constituent of caveolae can modulate Akt signaling pathway and enhances cell survival. Caveolin-1 was over-expressed on bladder cancer, prostate cancer, esophageal cancer and thyroid papillary carcinoma. The aim of this study is to investigate the expression of caveolin-1 on NPC and its impacts on clinical manifestations of NPC patients. The effect of caveolin-1 on Akt signaling in NPC-TW01 cells will be elucidated in this study. The expression of caveolin-1 was investigated in 74 NPC specimens, 29 control tissues and some cell lines. Our data revealed the caveolin-1 is over-expressed at transcription level in NPC patients (P=0.0002). Late-stage NPC (stage III, IV) patients have higher level of caveolin-1 expression than early-stage (stage I,II) ones (P=0.0002). After multiple regression analysis, three factors including tumor staging, tumor size (T) and lymph node metastasis (N) are included in the model with significant difference. The overall survival rate is poor in patients with high level of caveolin-1 expression (Log-rank test, P=0.03). Over-expressed caveolin-1 in TW01 cells increased more than 50% phosphorylated Akt protein intensity. Knockdown of caveolin-1 by four shRNAs substantially reduced Akt protein levels and almost abolished phosphorylated Akt protein compared with that in untreated TW01 and empty vector control cells. It suggested caveolin-1 may modulate Akt signaling in NPC TW01 cell lines.

黏蛋白型O-醣基化在大腸直腸癌進展和轉移中扮演之角色

Fri, 01 Jan 2016 00:00:00 GMT

標題: 黏蛋白型O-醣基化在大腸直腸癌進展和轉移中扮演之角色; Roles of mucin-type O-glycosylation in colorectal cancer progression and metastasis 作者: Ji-Shiang Hung; 洪基翔摘要: 背景及目的大腸直腸癌是全世界最常見及最致命的惡性疾病之一。雖然目前的治療方式已經顯著地延長第四期大腸直腸癌患者的存活，已經有遠端轉移的大腸直腸癌通常還是無法治癒。這個難題讓我們提出以下的疑問：目前全身性的大腸直腸癌治療方法，是否瞄準真正關鍵的腫瘤生長和轉移的分子機制？黏蛋白種類O-醣基化是蛋白質最常見的轉譯後修飾之一，並且和許多重要的生物功能有關。細胞表面分子異常的醣基化和細胞的惡性轉變相關。和腫瘤有關的Tn及T抗原屬於腫瘤胚胎醣類抗原，常見於大腸癌、乳癌、前列腺癌及軟巢癌組織中。這些腫瘤胚胎抗原極有潛力成為癌症診斷、影像檢查及治療的標的。然而，目前關於調控Tn及T抗原的醣化基因和其功能還不是瞭解的很清楚。這個研究重要的地方在於：（1）能幫助瞭解大腸直腸癌中異常的醣基因表現及其和病人預後的關係；（2）能闡明癌細胞的惡性表現是如何受到醣化基因的調控；以及（3）所得到的資訊可以幫助研發診斷及治療癌症的試劑與新藥。研究對象與方法 1. 為了要發現可以調控大腸癌細胞上T及Tn抗原的醣基因，我們用即時聚合酶鏈鎖反應來分析正常及癌化的大腸直腸組織裡不同的醣基因表現。找到有不同表現的醣基因後，再比較其表現量與病人預後的關聯性。那些和預後有相關的醣基因將會被選殖出來。 2. 為了確定是否這些選殖出來的醣基因和Tn及T抗原的表現有關，這些醣基因將會被轉殖到當腸直腸癌細胞株裡，然後以流式細胞儀分析癌細胞上Tn及T抗原的表現變化。此外，為了後續的實驗，我們選殖了這些基因並製作了其重組蛋白，並製造了這些基因的多株及單株抗體。 3. 為了研究這些醣基因的功能，我們使用了過度表現和siRNA敲除技術操作大腸癌細胞株後，以細胞增生、凋亡、粘附、遷移、侵襲和集落形成測定法來檢驗癌細胞的惡性表現程度。並進行動物實驗來了解這些醣基因對癌細胞在生物體內的轉移能力所造成的影響。結果 1. 來自台大醫院（NTUH）病人的配對組織以西方點墨法進行分析。與相鄰的非腫瘤組織相比，大腸直腸癌組織的C1GALT1有過度呈現的情況。C1GALT1的免疫組織化學分析結果指出，與正常的相對應組織相比，67.8％（59/87）的大腸直腸腫瘤有較高的C1GALT1呈現。此外，Kaplan-Meier生存分析顯示，與沒有C1GALT1過度呈現（T≤N）的大腸直腸癌病人相比，有C1GALT1過度呈現（T > N）的病人，其累積的存活率數據顯著地較低。另外，我們還觀察到，C1GALT1的呈現與T抗原的呈現有相關性，而後者是由PNA染色所檢測（圖6）。這些發現指出，C1GALT1的呈現在大腸直腸腫瘤中經常是過度呈現的情況，而其過度呈現與不佳的預後有相關性。 2. 在HCT116和SW480細胞中，C1GALT1的過度呈現會些微增加細胞的存活力，而減少C1GALT1則會些微抑制細胞存活力。transwell和matrigel侵襲實驗的結果顯示，在HCT116和SW480細胞，C1GALT1的過度呈現會顯著地增強細胞的遷移和侵襲。相反地，減少C1GALT1會抑制HCT116和SW620細胞的遷移和侵襲。這些發現指出，C1GALT1能調節大腸癌細胞的惡性行為。動物實驗的結果也顯示出，C1GALT1的過度表現會增加SW480轉移到肺部的數量，而抑制C1GALT1則會減少SW620轉移到肺部的數量。這個結果表示C1GALT1的表現能調控腫瘤在生物體內的生長和轉移能力。 3. 我們發現，C1GALT1可以調節bFGF所誘導的惡性表型。因為FGFR2在大腸直腸癌的意義已經明確地證實了，因此，我們研究了C1GALT1是否能調控FGFR2的醣基化和活動力。我們發現，在HCT116、SW480、和SW620細胞中，只有少量的內源性FGFR2會被凝集素PNA所減少，這指出，FGFR2上只有少量的Ť抗原。有趣的是，以神經氨酸酶處理後，PNA很容易就可以減少FGFR2，這指出，FGFR2帶有唾液酸T抗原。此外，我們發現，FGFR2也可以被凝集素VVA所減少，這指出，FGFR2上有Tn的存在。以神經氨酸酶去除唾液酸後，會增加凝集素VVA與FGFR2的結合，這指出，FGFR2上有唾液酸Tn的存在。這些結果強烈指出，在大腸癌細胞上，FGFR2上飾有O-聚醣。我們接下來研究，C1GALT1是否可以修飾FGFR2上的O-聚醣。在HCT116和SW480中，C1GALT1的過度呈現會降低VVA與FGFR2的結合；而在HCT116和SW620細胞中，減少C1GALT1會增加VVA與FGFR2的結合。這些結果指出，在大腸癌細胞中，C1GALT1能夠調控FGFR2上的O-聚醣結構。此外，在HCT116和SW480細胞中，C1GALT1的過度呈現會增加FGFR2和ERK 1/2的磷酸化，而此磷酸化是由bFGF誘導的。相反地，HCT116和SW620細胞中，在以bFGF處理後，減少C1GALT1會抑制FGFR2和ERK 1/2的磷酸化。這些結果指出，在大腸癌細胞中，C1GALT1能修飾FGFR2上的O-聚醣並調控bFGF所誘導的FGFR2活性。結論我們發現C1GALT1在大腸腫瘤中過度表現，且其過度表現與大腸直腸腫瘤病人的較差存活期有關。C1GALT1的過度表現會藉由修改FGFR2的O-醣基化來影響其活性，增強了大腸癌細胞侵襲的潛力和類幹細胞的特性。相反地，在體外和體內試驗中，C1GALT1的降低會抑制這些惡性特質。這些發現，對O-醣基化在大腸直腸癌中的相應角色開啟了全新的視野，並指出C1GALT1有望成為治療大腸直腸癌的治療標靶。; Background and objective Colorectal cancer is one of the most common and deadliest malignant disease worldwide. Although current treatment modalities have dramatically improved the survival of patients with stage IV colorectal cancer, colorectal cancers that spread to distant organs are usually not curable. This problem raises questions as to whether current systemic anti-colorectal cancer treatments are targeting molecular mechanisms that are truly critical to cancer growth and metastasis. Mucin-type O-glycosylation is one of the most common post-translational modifications of proteins and is associated with many important biological functions. Aberrant glycosylation of cell surface molecules is associated with malignant transformation. Tumor-associated Tn and T antigens are oncofetal carbohydrate antigens and present in colon, breast, prostate, and ovarian cancer. They are promising targets for cancer diagnosis, imaging, and therapeutics. However, the glycogenes responsible for regulating Tn and T antigens expression and their biological functions remain largely unknown. This research is significant in that: (1) it contributes to the understanding of altered glycogene expression in colorectal cancer and the association between their expression level and the patient prognosis; (2) it provides the knowledge about what and how the cancer cell behaviors are regulated by glycogenes; and (3) the gained information will allow us to develop diagnostic and therapeutic reagents for human cancers. Materials and methods 1. To identify novel glycogenes which can regulate Tn and T antigen expression in colorectal cancer cells, the differential expression of glycogenes in normal colorectal and cancerous tissues from colorectal cancer patients were analyzed by real-time RT-PCR. After the glycogenes with altered expression in colorectal cancer were found, the relation of expression level and the patient prognosis were examined. The glycogenes which have impact on patient’s survival were cloned. 2. To confirm whether these glycogenes can regulate Tn or T antigen expression, colon cancer cell lines were transfected with these genes. Then Tn and T antigen expression on cell surfaces was analyzed by flow cytometry. In addition, we cloned these genes and made their recombinant proteins as well as generated their polyclonal and monoclonal antibodies for subsequent experiments. 3. To investigate the function of these glycogenes in vitro, overexpression and siRNA knockdown techniques were used. Cell-based assays, including cell proliferation, apoptosis, adhesion, migration, invasion, and colony formation assays were performed. Animal study was also conducted to reveal the effect of the glycogenes on cancer metastasis in vivo. Results 1. Paired tissues from patients of the National Taiwan University Hospital (NTUH) were analyzed by Western blotting. C1GALT1 expression was found to be overexpressed in colorectal cancer tissues compared with adjacent non-tumor tissues. The results from immunohistochemistry of C1GALT1 indicated that 67.8% (59/87) of colorectal tumors showed higher C1GALT1 expression than their normal counterpart tissues. Moreover, a Kaplan-Meier survival analysis showed that the cumulative survival rate of colorectal cancer patients with C1GALT1 overexpression (T > N) was significantly lower than the patients without C1GALT1 overexpression (T ≤ N). In addition, we also observed that C1GALT1 expression is positively associated with T antigen expression as revealed by PNA staining. These findings suggest that C1GALT1 expression is frequently overexpressed in colorectal tumors and its overexpression is associated with poor survival. 2. Overexpression of C1GALT1 slightly increased cell viability in HCT116 and SW480 cells, whereas knockdown of C1GALT1 slightly inhibited cell viability in HCT116 and SW620 cells. We also analyzed migration and invasion by transwell and matrigel invasion assay, respectively. Results showed that overexpression of C1GALT1 significantly enhanced cell migration and invasion in HCT116 and SW480 cells. In contrast, knockdown of C1GALT1 suppressed cell migration and invasion in HCT116 and SW620 cells. These findings suggest that C1GALT1 can regulate malignant behaviors of colon cancer cells in vitro. We then conducted animal experiment and observed that overexpression of C1GALT1 increased lung metastasis of SW480 cells, whereas knockdown of C1GALT1 suppressed lung metastasis of SW620 cells. These results suggest that C1GALT1 expression is able to modulate tumor growth and metastasis in vivo. 3. We found that C1GALT1 can regulate bFGF-induced malignant phenotypes. Since the significance of FGFR2 in colorectal cancer has been clearly demonstrated, we investigated whether C1GALT1 can regulate FGFR2 glycosylation and activity. Only low amounts of endogenous FGFR2 were pulled down by PNA in HCT116, SW480, and SW620 cells, indicating small amounts of T antigens on FGFR2. Interestingly, after neuraminidase treatment, FGFR2 was easily pulled down by PNA, suggesting that FGFR2 carries sialyl T antigens. Moreover, FGFR2 could also be pulled down by VVA, indicating the presence of Tn on FGFR2. Removal of sialic acids with neuraminidase increased VVA binding to FGFR2, indicating the presence of sialyl Tn on FGFR2. These results strongly suggest that FGFR2 is decorated with O-glycans in colon cancer cells. We next investigated whether C1GALT1 can modify O-glycans on FGFR2. Overexpression of C1GALT1 decreased VVA binding to FGFR2 in HCT116 and SW480 cells, whereas knockdown of C1GALT1 increased VVA binding to FGFR2 in HCT116 and SW620 cells. These results suggest that C1GALT1 is able to modulate O-glycan structures on FGFR2 in colon cancer cells. Furthermore, overexpression of C1GALT1 increased bFGF-induced phosphorylation of FGFR2 and ERK1/2 in HCT116 and SW480 cells. Conversely, knockdown of C1GALT1 suppressed the phosphorylation of FGFR2 and ERK1/2 after bFGF treatment in HCT116 and SW620 cells. These findings suggest that C1GALT1 modifies O-glycans on FGFR2 and regulates bFGF-induced activation of FGFR2 in colon cancer cells. Conclusion We found that C1GALT1 is overexpressed in colorectal tumors and its overexpression is associated with poor survival of patients with colorectal tumors. C1GALT1 overexpression enhances the invasive potential and stem-like cell property of colon cancer cells via modifying O-glycosylation and activity of FGFR2. Conversely, C1GALT1 knockdown suppresses these malignant properties in vitro and in vivo. These findings open novel insights into the relevant role of O-glycosylation in colorectal cancer and suggest C1GALT1 as a promising therapeutic target for the treatment of colorectal cancer.